Cody L. Hoop

The Aggregation-Enhancing Huntingtin N-Terminus Is Helical in Amyloid Fibrils

The 17-residue N-terminus (htt(NT)) directly flanking the polyQ sequence in huntingtin (htt) N-terminal fragments plays a crucial role in initiating and accelerating the aggregation process that is associated with Huntingtons disease pathogenesis. Here we report on magic-angle-spinning solid-state NMR studies of the amyloid-like aggregates of an htt N-terminal fragment. We find that the polyQ portion of this peptide exists in a rigid, dehydrated amyloid core that is structurally similar to simpler polyQ fibrils and may contain antiparallel β-sheets. In contrast, the htt(NT) sequence in the aggregates is composed in part of a well-defined helix, which likely also exists in early oligomeric aggregates. Further NMR experiments demonstrate that the N-terminal helical segment displays increased dynamics and water exposure. Given its specific contribution to the initiation, rate, and mechanism of fibril formation, the helical nature of htt(NT) and its apparent lack of effect on the polyQ fibril core structure seem surprising. The results provide new details about these disease-associated aggregates and also provide a clear example of an amino acid sequence that greatly enhances the rate of amyloid formation while itself not taking part in the amyloid structure. There is an interesting mechanistic analogy to recent reports pointing out the early-stage contributions of transient intermolecular helix-helix interactions in the aggregation behavior of various other amyloid fibrils.

β-Hairpin-Mediated Nucleation of Polyglutamine Amyloid Formation

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntingtons disease. Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core.

Significance Huntington’s disease is a devastating and incurable inherited neurodegenerative disease. Like at least eight other diseases, its primary genetic cause is the CAG repeat expansion in a specific gene. Mutant huntingtin protein undergoes misfolding and aggregation, causing degeneration of neurons through as-yet poorly understood mechanisms. Attempts to characterize the implicated protein deposits have until now had limited success. We present our structural studies of mutant huntingtin-derived protein deposits by advanced solid-state NMR spectroscopy. We determine the essential structural features of the fibrils’ rigid core, which is shown to feature intramolecular β-hairpins tied together via interdigitating extended side chains. These structural insights have direct implications for the mechanism by which the mutant protein misfolds and self-assembles. Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

Serine phosphorylation suppresses huntingtin amyloid accumulation by altering protein aggregation properties.

Aggregation of expanded polyglutamine repeat-containing fragments of the huntingtin (htt) protein may play a key role in Huntingtons disease. Consistent with this hypothesis, two Ser-to-Asp mutations in the 17-amino-acid N-terminal htt(NT) segment abrogate both visible brain aggregates and disease symptoms in a full-length Q(97) htt mouse model while compromising aggregation kinetics and aggregate morphology in an htt fragment in vitro [Gu et al. (2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron64, 828-840]. The htt(NT) segment has been shown to play a critical role in facilitating nucleation of amyloid formation in htt N-terminal exon1 fragments. We show here how these Ser-to-Asp mutations dramatically affect aggregation kinetics and aggregate structural integrity. First, these negatively charged Ser replacements impair the assembly of the α-helical oligomers that play a critical role in htt amyloid nucleation, thus providing an explanation for reduced amyloid formation rates. Second, these sequence modifications alter aggregate morphology, decrease aggregate stability, and enhance the steric accessibility of the htt(NT) segment within the aggregates. Together, these changes make the sequence-modified peptides kinetically and thermodynamically less likely to aggregate and more susceptible, if they do, to posttranslational modifications and degradation. These effects also show how phosphorylation of a protein might achieve cellular effects via direct impacts on the proteins aggregation properties. In fact, preliminary studies on exon1-like molecules containing phosphoryl-Ser residues at positions 13 and 16 show that they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser-to-Asp mutants.

Structural Changes and Proapoptotic Peroxidase Activity of Cardiolipin-Bound Mitochondrial Cytochrome c.

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly (13)C,(15)N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Polyglutamine amyloid core boundaries and flanking domain dynamics in huntingtin fragment fibrils determined by solid-state nuclear magnetic resonance

In Huntington’s disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A “protective” C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (httNT) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the β-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt β-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the httNT α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

STRUCTURAL CHARACTERIZATION OF THE CAVEOLIN SCAFFOLDING DOMAIN IN ASSOCIATION WITH CHOLESTEROL-RICH MEMBRANES

Members of the caveolin protein family are implicated in the formation of caveolae and play important roles in a number of signaling pathways and in the regulation of various proteins. We employ complementary spectroscopic methods to study the structure of the caveolin scaffolding domain (CSD) in caveolin-1 fragments, while bound to cholesterol-rich membranes. This key domain is thought to be involved in multiple critical functions that include protein recognition, oligomerization, and cholesterol binding. In our membrane-bound peptides, residues within the flanking intramembrane domain (IMD) are found to adopt an α-helical structure, consistent with its commonly believed helical hairpin conformation. Intriguingly, in these same peptides, we observe a β-stranded conformation for residues in the CSD, contrasting with earlier reports, which commonly do not reflect β-structure. Our experimental data based on solid-state NMR, CD, and FTIR are found to be consistent with computational analyses of the secondary structure preference of the primary sequence. We discuss how our structural data of membrane binding Cav fragments may match certain general features of cholesterol-binding domains and could be consistent with the role for CSD in protein recognition and homo-oligomerization.

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in “domain-swapping” fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.